Abstract

Organization of lipids into membrane microdomains is a vital mechanism of protein processing. Here we show that overexpression of ERG6, a gene involved in ergosterol synthesis, elevates sterol levels 1.5-fold on the vacuole membrane and enhances their homotypic fusion. The mechanism of sterol-enhanced fusion is not via more efficient sorting, but instead promotes increased kinetics of fusion subreactions. We initially isolated ERG6 as a suppressor of a vrp1Delta growth defect selective for vacuole function. VRP1 encodes verprolin, an actin-binding protein that colocalizes to vacuoles. The vrp1Delta mutant has fragmented vacuoles in vivo and isolated vacuoles do not fuse in vitro, indicative of a Vrp1p requirement for membrane fusion. ERG6 overexpression rescues vrp1Delta vacuole fusion in a cytosol-dependent manner. Cytosol prepared from the vrp1Delta strain remains active; therefore, cytosol is not resupplying Vrp1p. Las17p (Vrp1p functional partner) antibodies, which inhibit wild-type vacuole fusion, do not inhibit the fusion of vacuoles from the vrp1Delta-ERG6 overexpression strain. Vacuole-associated actin turnover is decreased in the vrp1Delta strain, but recovered by ERG6 overexpression linking sterol enrichment to actin remodeling. Therefore, the Vrp1p/Las17p requirement for membrane fusion is bypassed by increased sterols, which promotes actin remodeling as part the membrane fusion mechanism.

Identification of ERG6 as a suppressor of the vrp1Δ strain. Colony growth assays using a 10-fold dilution series of strains are shown in the BY4742 (WT) background. (A) Addition of 6 mM caffeine to YPD selectively impairs growth of mutants involved in vacuole fusion such as vam3Δ (KTY14), vps33Δ (KTY11), and ypt7Δ (KTY12), but not the vps5Δ (KTY13) mutant, which is not involved in fusion. Growth of the actin regulatory mutant, vrp1Δ (KTY10), is also impaired on YPD-caffeine. (B) Growth of the vrp1Δ mutant is recovered when transformed with a YEp13 genomic library containing the VRP1, LAS17, or ERG6 gene (strains KT10-0, 10-40, 10-16, respectively). The table on the right summarizes the suppressor analysis. (C) Transformation with a high-copy plasmid (2μ) containing the ERG6 opening reading frame rescues growth of the vrp1Δ but not vps33Δ strains on YPD-caffeine (left panel). Multiple ERG6 copies also suppress YPD-(1.8 M) sorbitol-sensitive growth (right panel). Transformation with a low-copy plasmid (cen) does not rescue growth.

Characterization of ERG6 overexpression strains. The galactose-regulated GAL1 promoter and three HA epitopes were introduced inframe at the 5′ end of the ERG6 gene. (A) Expression of PGAL1-HA-ERG6 was examined 24 h after inoculating 10 ml of YP media with varying dextrose:galactose ratios from a single YPD preculture of strain KTY3. Strain KTY1 was included as a negative control (no PGAL1-HA-ERG6 allele). Equivalent culture volumes were processed for immunoblot analysis with anti-HA and anti-actin as a load control. Bands were analyzed by densitometry and normalized to 1% dextrose to calculate fold induced. (B) Erg6p expression levels were examined in cultures grown in YP 1% dextrose/1% galactose to 1 OD600 for vacuole isolation. Culture equivalents of 0.25 OD600 units were processed from strains with PGAL1-HA-ERG6 induction, KTY3-4 (WT) and KTY7-8 (vrp1Δ), and from cultures without PGAL1-HA-ERG6, KTY1-2 (WT) and KTY5-6 (vrp1Δ). Immunoblot analysis was performed with both anti-Erg6p (gift from G. Daum) and anti-HA for direct comparison. Anti-Erg6p bands were analyzed by densitometry and normalized to wild-type pep4Δ (KTY1). (C) Growth curves were generated for wild-type (strains KTY1-4) and vrp1Δ mutants (strains KTY5-8) by inoculating 0.1 ml of 1 OD600 cultures into 50 ml of fresh YP media containing either 2% dextrose (top panel), 2% galactose (middle panel), or 1% dextrose/1% galactose (bottom panel).

ERG6 suppresses a specific growth defect of the las17-16 mutant, but not that of other mutants downstream of Vrp1p/Las17p. Colony growth assays using a 10-fold dilution series of cultures are shown with their respective parental (WT) and mutant backgrounds (see Table 1). Addition of 1.8 M sorbitol to YPD selectively impairs the growth of mutants involved in vacuole biogenesis (Koning et al., 2002) as well as the actin-remodeling mutants las17-16 (A), arc35-1 (B), and the direct actin mutants act1-157 and act1-101 (C). Transformation with the ERG6 gene on a multicopy plasmid (pERG6–2μ) restores growth of the las17-16 mutant (A), but has no effect on the other mutants (B and C).

ERG6 induction increases nystatin sensitivity. (A) Colony growth assays to test the sensitivity of strains to 10 μg/ml nystatin, indicative of increased levels of ergosterol. A 10-fold dilution series of the indicated strains were plated on YP media with 2% dextrose (YPD) or 1% galactose/0.2% dextrose (YPG). Growth was examined after 2 d in the absence of nystatin and 4 d in the presence of nystatin.

Release of vacuole-associated actin requires ATP and Vrp1p. Standard fusion reactions were analyzed for bound actin by coprecipitation with vacuoles and immunoblot analysis. At the specified times, reactions were stopped by fivefold dilution on ice and immediate centrifugation to precipitate vacuoles and associated proteins. Changes in vacuole-associated actin were determined by densiometric analysis of immunoblots. (A) Immunoblots showing typical actin reduction using vacuoles from wild-type and vrp1Δ strains, either with (PGAL1-ERG6) or without sterol enrichment. Actin release activity is not blocked by incubation with 250 μg/ml Sec17p antibodies (αS17) but is block by addition of 0.025 U/ml apyrase VII (Apy). (B) Quantification of release activity by analysis of vacuole-associated actin remaining after 60 min using reactions as in panel A. Immunoblots from five experiments were analyzed by densitometry (see Materials and Methods), and values represent the average reduction of vacuole-associated actin using 0 min as 100% for each data set. Error bars, SE.